Vertebrate craniofacial development is a complex process involving communication between multiple tissues and cell types. Neural crest cells form most of the skeletal structures and tendons of the head, and pattern the mesoderm-derived head muscles. How tendon serves to integrate the musculoskeletal system is unknown because we know almost nothing about the genetics and cellular interactions underlying tendon development. The movements and functions of the human head depend on 150 individual muscles, and loss of tendon or of tendon-muscle interactions has been implicated in human craniofacial syndromes with muscle defects. Similarly, preliminary evidence shows that zebrafish cyp26b1 mutants have craniofacial muscle defects and lack differentiated tendon in the head. Preliminary studies with a scxa morpholino show that inhibition of tendon differentiation disrupts craniofacial muscles. The objective of the proposed research is to characterize the role of tendon in integrating the zebrafish craniofacial musculoskeletal system. The first specific aim will examine the function of cyp26b1 and scxa in tendon differentiation by A) neural crest cell transplantation, B) gene expression analysis, and C) comparison of mutant phenotypes. The second specific aim will characterize the integration of the head musculoskeletal system in cyp26b1 and scxa mutants by A) scxa lineage mapping and b) in vivo time-lapse imaging of developing tendon and muscle. The proposed research will yield new tools and insights for musculoskeletal research and characterize for the first time in vivo interactions between tendon and muscle tissue. These results will uncover mechanisms of craniofacial integration, an understudied aspect of human craniofacial development.